Ptdlns(3)-kinases play essential roles in signal transduction and membrane trafficking, and act by catalyzing the formation of 3'-phosphoinositides. Impairment in their function leads to diseases such as cancer and type 2 diabetes, and thus identifying the direct effectors of 3'-phosphoinositides is crucial to our progress in ameliorating these diseases. Work from our laboratory demonstrated that a specific protein motif, the FYVE domain, binds with high affinity and specificity to Ptdlns(3)P, a 3'-phosphoinositide highly enriched in endosomes (Patki et al. (1998), Nature 394: 433). Approximately 40 proteins in the human genome contain FYVE domains, implicating them as candidates for important functions in signal transduction and membrane trafficking. Thus for example, the FYVE-domain containing protein SARA is required for TGFp signaling to Smad2, which occurs in early endosomes (Hayes et al. (2003) J. Cell. Biol. 158(7): 1239-49. To accelerate the identification of the functions of FYVE domain-containing proteins, we have used siRNA screens in C. elegans. Using a strain that reveals endocytosis defects, we have identified a highly conserved, previously uncharacterized protein essential for endocytosis, called WDFY2. Its mammalian homologue is expressed ubiquitously, and its depletion from cultured cells results in a pronounced inhibition of transferrin uptake. Moreover, WDFY2 is found on vesicles that reside within 100 nm from the plasma membrane, and which lack typical early endocytic markers, thus defining a previously unrecognized class of early endosomes. Furthermore, depletion of WDFY2 inhibits insulin- stimulated glucose uptake in 3T3-L1 adipocytes to an extent similar to that seen upon depletion of the serine-threonine kinase Akt2. A direct interaction between WDFY2 and Akt has been observed, suggesting that WDFY2 may have a unique, conserved dual role in signal transduction and membrane trafficking. Because of its potentially central role in these essential processes in mammalian cells, in this competing proposal we will focus on understanding the function and mechanism of action of WDFY2. We seek to: 1) Define the molecular composition of WDFY2-enriched endosomes using immuno-isolation techniques and mass spectrometry. 2) Define the mechanism by which depletion of WDFY2 leads to inhibition of endocytosis, by analyzing the kinetics of internalization and recycling of the transferrin receptor. 3) Define the function of WDFY2 in insulin-stimulated glucose uptake in adipocytes, by testing whether insulin activation of Akt2 is impaired, or whether GLUT4 trafficking is impaired in the absence of WDFY2. 4) Define the mechanism of action of WDFY2 by identifying proteins that specifically interact with its WD-40 motifs using proteomic and 2-hybrid approaches.